Method for obtaining functionally enhanced mitochondria and use thereof

Abstract

The present invention relates to a method for obtaining a functionally enhanced mitochondria and a use thereof, and specifically, to: a mitochondria isolated from cells overexpressing glutathione synthase; and a use thereof for preventing, alleviating, or treating muscle weakness-related diseases.

Description

Method for obtaining function-enhanced mitochondria and uses thereof

[0001] The present invention relates to a method for obtaining mitochondria with enhanced function and the use thereof, specifically to mitochondria isolated from cells in which glutathione synthase is overexpressed and the use thereof for the prevention, improvement, or treatment of diseases related to muscle weakness.

[0002] This application claims priority based on Korean Patent Application No. 10-2024-0173071 filed on November 28, 2024, and all contents disclosed in the specification and drawings of said application are incorporated into this application.

[0003] Skeletal muscle is the largest organ in the human body, accounting for 40 to 50% of total body weight, and plays a crucial role in various metabolic functions, including energy homeostasis and heat generation. As the human body ages, its composition changes, leading to a redistribution of body fat and protein. Around the age of 50, the rate of protein synthesis within muscle cells slows down compared to the rate of breakdown, causing muscles to begin degenerating rapidly. Sarcopenia refers to a condition in which a person's body mass decreases by approximately 13–24% of their normal weight, characterized by a decline in protein content, fiber diameter, muscle strength production, and fatigue resistance. Sarcopenia is caused by various factors, including sepsis, cancer, renal failure, excess glucocorticoids, neuropathy, muscle disuse, obesity, and the aging process. The primary causes cited for this condition include the abnormally rapid decline in the number of muscle stem cells, their differentiation potential, the quantity of skeletal muscle, and muscle quality that occurs as aging progresses. In October 2016, the World Health Organization (WHO) assigned a new disease classification code to sarcopenia, officially classifying it as a new geriatric disease. While the prevalence of sarcopenia is 13–24% in people under 70, it increases sharply to over 50% in those over 80. However, there are currently no treatments demonstrating a clear therapeutic effect for sarcopenia, and the current situation is limited to preventive measures such as strength training and protein intake. Consequently, there is a continuously emerging need for the development of drugs effective for treating sarcopenia, which is on the rise due to the aging of the population. Meanwhile, sarcopenic obesity, characterized by an increase in body fat alongside a decrease in muscle mass and strength due to aging, is also becoming a problem.It is estimated that obesity and sarcopenia in the elderly have a synergistic effect, exacerbating not only the risk of functional and metabolic disorders but also the risk of death, and it is predicted that they interact strongly from an etiological perspective. Therefore, in the case of overweight or obese individuals, or those of normal weight but with high body fat mass, increasing muscle mass and decreasing body fat increases basal metabolic energy, which can improve, prevent, and treat metabolic diseases such as diabetes and hypertension (Republic of Korea Registered Patent 10-2507494).

[0004] Recently, cell therapy products for sarcopenia have been actively researched. Cell therapy is a technology that induces the regeneration of damaged tissue, the regulation of immune responses, or the supplementation of deficient cellular functions by administering cells engineered in vitro to patients; it is known to offer the potential for a fundamental cure compared to conventional chemical drug treatments. However, these cell therapies still harbor numerous technical limitations, which imposes various constraints on their clinical application. Due to low engraftment and survival rates of cells in vivo after administration, the therapeutic effects are temporary and reproducible. Furthermore, in the case of live cell therapies, potential risks of immunogenicity and tumorigenicity still persist. Additionally, compared to general chemical drugs, the manufacturing process is highly complex, resulting in very high production costs and making mass production difficult. Moreover, because the degree of differentiation and metabolic status of cells change during the culture process, functional differences arise even within the same cell population, making it difficult to ensure quality consistency between products.

[0005] Therefore, there is an urgent need to develop an effective alternative treatment for sarcopenia that is neither a cell therapy nor a general chemical drug.

[0006] The present invention was devised to solve the problems of the prior art as described above. The inventors conducted diligent research to develop a novel mitochondrial transplant therapy (MitoTherapy) instead of a cell therapy, and confirmed that mitochondria derived from cells overexpressing glutathione synthetase exhibited significant effects in the regeneration of reduced muscle, recovery of damaged muscle, and / or improvement of muscle function, thereby completing the present invention.

[0007] The present invention provides cell-derived mitochondria that overexpress glutathione synthetase.

[0008] In addition, the present invention provides a mitochondrial transplant therapy (MitoTherapy) for the prevention or treatment of muscle weakness-related diseases, comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient.

[0009] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of diseases related to muscle weakness, comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient.

[0010] In addition, the present invention provides a food composition for the prevention or improvement of diseases related to muscle weakness, comprising cell-derived mitochondria that overexpress glutathione synthetase as an active ingredient.

[0011] In addition, the present invention provides a method for preventing or treating a muscle weakness-related disease, comprising the step of administering a composition containing cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient in a therapeutically effective amount to an individual in need.

[0012] In addition, the present invention provides a use for a composition comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient for the prevention, improvement, or treatment of diseases related to muscle weakness.

[0013] In addition, the present invention provides a use for producing a drug for the prevention, improvement, or treatment of muscle weakness-related diseases, comprising a composition containing cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient.

[0014] In addition, the present invention provides an antioxidant composition comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient.

[0015] In addition, the present invention provides an antioxidant method comprising the step of administering a composition containing cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient to an individual in need of the composition in a therapeutically effective amount.

[0016] In addition, the present invention provides a use for an antioxidant composition comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient.

[0017] In addition, the present invention provides an anti-inflammatory composition comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient.

[0018] In addition, the present invention provides a method for reducing inflammation comprising the step of administering a composition containing cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient in a therapeutically effective amount to an individual in need.

[0019] In addition, the present invention provides a use for anti-inflammatory purposes of a composition comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient.

[0020] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of mitochondrial dysfunction comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient.

[0021] In addition, the present invention provides a method for treating mitochondrial dysfunction comprising the step of administering a composition containing cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient in a therapeutically effective amount to an individual in need.

[0022] In addition, the present invention provides a composition comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient for the prevention or treatment of mitochondrial dysfunction.

[0023] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0024] The present invention provides cell-derived mitochondria that overexpress glutathione synthetase.

[0025] In addition, the present invention provides a mitochondrial transplant therapy (MitoTherapy) for the prevention or treatment of muscle weakness-related diseases, comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient.

[0026] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of diseases related to muscle weakness, comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient.

[0027] In addition, the present invention provides a food composition for the prevention or improvement of diseases related to muscle weakness, comprising cell-derived mitochondria that overexpress glutathione synthetase as an active ingredient.

[0028] In addition, the present invention provides a method for preventing or treating a disease related to muscle weakness, comprising the step of administering a composition containing cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient in a therapeutically effective amount to an individual in need thereof. The term “administration” is used with the same meaning as “mitochondria transplantation.”

[0029] In addition, the present invention provides a use for a composition comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient for the prevention, improvement, or treatment of diseases related to muscle weakness.

[0030] In addition, the present invention provides a use for producing a drug for the prevention, improvement, or treatment of muscle weakness-related diseases, comprising a composition containing cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient.

[0031] In addition, the present invention provides an antioxidant composition comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient.

[0032] In addition, the present invention provides an antioxidant method comprising the step of administering a composition containing cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient to an individual in need of the composition in a therapeutically effective amount. The antioxidant method is a method for reducing reactive oxygen species (ROS) in the body.

[0033] In addition, the present invention provides an antioxidant use of a composition comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient. The antioxidant use is to reduce reactive oxygen species (ROS) in the body.

[0034] In addition, the present invention provides an anti-inflammatory composition comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient.

[0035] In addition, the present invention provides a method for reducing inflammation comprising the step of administering a composition containing cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient in a therapeutically effective amount to an individual in need.

[0036] In addition, the present invention provides a use for anti-inflammatory purposes of a composition comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient.

[0037] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of mitochondrial dysfunction comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient.

[0038] In addition, the present invention provides a method for treating mitochondrial dysfunction comprising the step of administering a composition containing cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient in a therapeutically effective amount to an individual in need.

[0039] In addition, the present invention provides a composition comprising cell-derived mitochondria overexpressing glutathione synthetase as an active ingredient for the prevention or treatment of mitochondrial dysfunction.

[0040] In one embodiment of the present invention, the mitochondria satisfy one or more of the following characteristics: i) exhibiting a high glutathione concentration; ii) exhibiting a high membrane potential; iii) exhibiting high resistance to oxidative stress; and iv) increasing muscle mass. The term “high” means significantly higher compared to mitochondria derived from the same ungenetically modified cell line cultured under the same conditions. The high membrane potential can be confirmed by the fact that the JC-1 polymer is greater than the JC-1 monomer.

[0041] In another embodiment of the present invention, the cell overexpressing glutathione synthase is a cell transformed with a recombinant expression vector comprising a promoter and a polynucleotide encoding glutathione operably linked thereto, wherein “overexpression” refers to an increased expression level of glutathione synthase compared to the same ungenetically modified cell line cultured under the same conditions. The recombinant expression vector must be prepared in a form that can be expressed in the human body, and preferably may include a promoter, enhancer, start codon, stop codon, polyadenylation signal, etc., but is not limited thereto as long as it is a form known to be expressible in the body. The “derived” refers to something isolated from the cell overexpressing glutathione synthase and is used with the same meaning as “mitochondria isolated from the cell overexpressing glutathione synthase.” Most preferably, it refers to mitochondria derived from human embryonic stem cell-derived mesenchymal stem cells (hES-MSC) transformed with a recombinant expression vector comprising a polynucleotide encoding a promoter and a glutathione operably linked thereto. Or it refers to mitochondria isolated from human embryonic stem cell-derived mesenchymal stem cells (hES-MSC) transformed with a recombinant expression vector comprising a polynucleotide encoding a promoter and a glutathione operably linked thereto.

[0042] In another embodiment of the present invention, the cell may be a somatic cell or a stem cell, and the stem cell may be one or more selected from the group consisting of mesenchymal stem cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, and induced pluripotent stem cells. The mesenchymal stem cell may be a bone marrow-derived mesenchymal stem cell (BM-MSC), an adipose tissue-derived mesenchymal stem cell (AD-MSC), a cord blood-derived mesenchymal stem cell (UC-MSC), a cord tissue-derived mesenchymal stem cell (Wharton's jelly-MSC), a placenta-derived stem cell (pMSC), a tooth-derived mesenchymal stem cell (DP-MSC), etc. Most preferably, the cell is a human embryonic stem cell-derived mesenchymal stem cell (hES-MSC).

[0043] In another embodiment of the present invention, diseases related to muscle weakness are diseases caused by mitochondrial dysfunction, and may include all diseases resulting from the decline in function of muscle cells or nerve cells caused by a decrease in ATP production due to mitochondrial dysfunction. The mitochondria of the present invention can be used to prevent, improve, or treat diseases caused by the decline in mitochondrial function by replacing mitochondria with reduced function and through functions such as improving ATP production capacity and increasing antioxidant capacity. In another embodiment of the present invention, the muscle weakness-related disease is one or more selected from the group consisting of sarcopenia, muscular atrophy, muscle dystrophy, cardiac atrophy, mitochondrial myopathy, amyotrophic lateral sclerosis (ALS), cardiomyopathy, Parkinson's disease, Huntington's disease, Alzheimer's disease, and peripheral neuropathy. Most preferably, the disease is sarcopenia, muscular atrophy, mitochondrial myopathy, amyotrophic lateral sclerosis, or cardiac atrophy. When administered, the above mitochondria are internalized into cells within the body, and can treat muscle weakness-related diseases by increasing muscle mass and strengthening muscle power through restoring or regenerating muscles in individuals with muscle weakness-related diseases, that is, individuals with reduced or damaged muscles, or by increasing mitochondrial function, or by increasing resistance to oxidative stress.

[0044] In another embodiment of the present invention, the glutathione synthase may comprise the amino acid sequence represented by SEQ ID NO. 2. Most preferably, the glutathione synthase is a human glutathione synthase. Additionally, variants of SEQ ID NO. 2 are included within the scope of the present invention. Specifically, the glutathione synthase may comprise an amino acid sequence having sequence homology of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more with respect to the amino acid sequence of SEQ ID NO. 2. Most preferably, the glutathione synthase may comprise an amino acid sequence having sequence homology of 98% or more. The “% of sequence homology” is determined by comparing the optimally arranged sequence with a comparison region, wherein a portion of the polynucleotide sequence or amino acid sequence in the comparison region may include additions or deletions (i.e., gaps) compared to a reference sequence (without additions or deletions) for the optimal arrangement of the sequence. However, it is not limited to any type known as a human glutathione synthase. Alignment performed for the purpose of determining the percentage of sequence identity can be achieved in various ways within the common sense of a person skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. A person skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0045] Mitochondria derived from cells overexpressing glutathione synthase according to the present invention exhibit significantly higher glutathione concentration, higher membrane potential, and higher resistance to oxidative stress compared to mitochondria derived from the same ungenetically modified cell line cultured under the same conditions. Furthermore, when administered to damaged or reduced muscle, they can increase muscle mass and improve muscle strength. Additionally, the increase in glutathione concentration and the decrease in 4NHE when mitochondria according to the present invention are administered or transplanted demonstrate a powerful antioxidant effect, indicating that they can restore the function of damaged mitochondria to a normal level and improve redox balance. Moreover, mitochondria according to the present invention possess an anti-inflammatory effect against inflammation caused by aging. Therefore, mitochondria according to the present invention are expected to be widely applicable to diseases related to muscle damage or muscle weakness caused by various factors.

[0046] FIG. 1A is a diagram briefly illustrating a method for isolating mitochondria according to one embodiment of the present invention, and FIG. 1B is a diagram showing the result of observing isolated mitochondria by fluorescence according to one embodiment of the present invention.

[0047] Figure 2 is a diagram showing the results of confirming the glutathione levels of mitochondria isolated from MSCs transformed with a GS expression vector according to one embodiment of the present invention.

[0048] FIG. 3A is a diagram showing the results of confirming the effect of oxidative stress on mitochondria according to one embodiment of the present invention using a fluorescence microscope, and FIG. 3B is a diagram showing the results confirmed using TEM.

[0049] FIG. 4A is a diagram briefly illustrating a method for testing the efficacy of mitochondria delivered into a cell and transplanted mitochondria according to one embodiment of the present invention, and FIG. 4B is a diagram showing the results of confirming the mitochondria delivered into the cell by fluorescence.

[0050] Figure 5 is a figure showing the results of confirming the recovery and / or regenerative ability of muscles damaged by oxidative stress of mitochondria according to one embodiment of the present invention by staining the myotubes and nuclei using immunofluorescence.

[0051] FIG. 6A is a figure showing the results of confirming the recovery and / or regenerative ability of damaged muscles of Mock-mito and GS-mito according to one embodiment of the present invention by staining the root canal and nucleus using immunofluorescence, and FIG. 6B is a figure showing the results of confirming the results of staining the root canal with fluorescence.

[0052] FIG. 7 shows a 1.3mm according to an embodiment of the present invention. 2 Number of root canals contained within (A), 1.3 mm 2 This is a diagram showing the quantification of the percentage of the total area of ​​Fast MyHC positive (+) areas contained within (B), the fusion index % (C), and the ratio of length to width of the root canal (D).

[0053] FIG. 8A is a diagram briefly illustrating an experimental method for confirming mitochondrial function in transplanted cells according to an embodiment of the present invention as an oxygen consumption rate, FIG. 8B is a diagram showing the results of observing the morphology of a cell with transplanted mitochondria under a microscope, FIG. 8C is an example diagram of the oxygen consumption rate measurement results, and FIG. 8D and 8E are diagrams showing the results of confirming the measured oxygen consumption rate.

[0054] FIG. 9 is a diagram briefly illustrating an experimental method for confirming the therapeutic effect on muscle loss in vivo according to one embodiment of the present invention.

[0055] FIG. 10 is a diagram showing experimental results confirming muscle growth ability according to one embodiment of the present invention.

[0056] FIG. 11 is a diagram showing experimental results confirming the degree of functional recovery of muscles according to one embodiment of the present invention.

[0057] FIG. 12 is a diagram showing the results of confirming the morphology of muscle tissue and mitochondria according to one embodiment of the present invention using TEM.

[0058] FIG. 13 is a diagram showing experimental results confirming the degree of functional recovery of muscles according to one embodiment of the present invention.

[0059] FIG. 14 is a diagram showing experimental results confirming muscle growth ability according to one embodiment of the present invention.

[0060] FIG. 15 is a diagram showing experimental results confirming the degree of functional recovery of muscles according to one embodiment of the present invention.

[0061] FIG. 16 is a diagram showing the experimental results of confirming the glutathione concentration in serum according to one embodiment of the present invention.

[0062] FIG. 17 is a diagram showing experimental results confirming the antioxidant effect according to one embodiment of the present invention.

[0063] FIG. 18 is a diagram showing experimental results confirming the anti-inflammatory effect according to one embodiment of the present invention.

[0064] In all claims below, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0065] In all claims below, the terms of degree, such as “about,” “approximately,” “substantially,” “average,” “generally,” etc., are used in the sense of being at or close to the value when inherent manufacturing and material tolerances are presented in the sense mentioned, and are used to prevent unscrupulous infringers from unfairly exploiting the disclosure regarding the precise or absolute value mentioned to aid in understanding the present invention. For example, the terms “about,” “approximately,” “substantially,” “average,” “generally,” etc., may refer to amounts within 10%, within 5%, within 3%, within 1%, within 0.1%, and within 0.01% of the mentioned amount.

[0066] Additionally, in the entire claim below, "step of" or "step of" does not mean "step for".

[0067] In all claims below, the term “group consisting of” or “combination thereof” included in a Markush-type expression means one or more mixtures or combinations selected from a group consisting of components described in a Markush-type expression, and means including one or more selected from the group consisting of said components.

[0068] In the entirety of the following claims, the description of "A and / or B" means "A or B, or A and B".

[0069] In the entirety of the following claims, the term "active ingredient" refers to any substance used interchangeably with active drugs, active ingredients, active formulations, drugs, and therapeutic formulations, and used to prevent, alleviate, improve, or treat a target disease.

[0070] In this specification, including all claims below, the term “subject” refers to a target to which the composition of the present invention may be administered, preferably a mammal including humans, and may include, for example, human or non-human primates, mice, rats, non-human animals such as dogs, cats, horses, cattle, and pigs, prokaryotic cells, isolated eukaryotic cells, isolated mammalian tissues, eukaryotic organisms other than humans, but there is no limitation on the subject. Furthermore, the term “subject in need thereof” may preferably refer to a subject having a disease related to muscle weakness or at risk of a disease related to muscle weakness and requiring prevention or treatment, but is not limited thereto. Alternatively, it may preferably refer to a subject with an increased inflammatory response or at risk of inflammatory disease resulting therefrom. Alternatively, it may preferably refer to a subject with increased oxidative stress or at risk of disease caused by oxidative stress.

[0071] Including all claims below, the pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceuticalally effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0072] In all claims below, the term “isolated” means altered or removed from its natural state. For example, mitochondria naturally present under normal conditions in living cells are not “isolated,” whereas the mitochondria of the present invention are “isolated” by being partially or completely separated from coexisting materials under natural conditions. The isolated mitochondria may exist in a substantially purified form or may exist in a non-natural environment, such as a host cell.

[0073] Including all claims below, the term "administration" in this specification means providing a specific composition of the present invention to an individual by any appropriate method.

[0074] In this specification, including all claims below, "prevention" refers to any act of suppressing or delaying the onset of a target disease, and preferably includes both primary prevention, which prevents the onset beforehand, and secondary prevention, which detects the onset early and treats it in a timely manner. In addition, in this specification, including all claims below, "treatment" refers to any act of improving or beneficially altering a target disease and associated metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and "improvement (or amelioration)" refers to any act of reducing parameters related to the target disease, such as the severity of symptoms, by administering a composition according to the present invention. More specifically, this refers to any indicator indicating success in the treatment of a pathological condition or disease, and includes not only objective or subjective parameters such as abatement, remission, or diminishing of symptoms, but also improvement in the patient's physical or mental health status. Symptom improvement (amelioration) may be based on objective or subjective parameters, including the results of physical examination and / or psychiatric evaluation.

[0075] In this specification, including all claims below, “glutathione synthetase” is an enzyme that produces glutathione, that is, a crystalline peptide composed of glutamic acid, cysteine, and glycine. There are no limitations on the glutathione synthetase known to exist in the human body, but preferably it may include the amino acid sequence represented by SEQ ID NO. 2. Additionally, variants of SEQ ID NO. 2 are included within the scope of the present invention. Specifically, it may include an amino acid sequence having sequence homology of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more with the amino acid sequence of SEQ ID NO. 2. The variant may include conservative amino acid substitutions. Conservative amino acid substitution refers to replacing an amino acid with another amino acid that preserves the physicochemical characteristics of the polypeptide (e.g., D can be replaced with E or vice versa, N with Q, or L or I with V or vice versa). Therefore, the substituting amino acid generally has characteristics similar to the amino acid to be replaced, such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains, etc. Isomers of natural L-amino acids, such as D-amino acids, may be included. Additionally, the glutathione synthase may be encoded from the nucleotide sequence represented by SEQ ID NO. 1 or a variant thereof. The variant may include a nucleotide sequence having sequence homology of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more with the nucleotide sequence of SEQ ID NO. 1. Variants of the above base sequence also include those in which the nucleotide sequence has been altered due to "degeneracy of the genetic code." "Degeneracy of the genetic code" means that there are multiple different codons that code for a single amino acid.For example, in the case of the amino acid leucine, it is encoded identically by various codons such as "CUU", "CUC", "CUA", "CUG", and "UUA", and this is called the degenerate nature of the genetic code. That is, the nucleic acid sequence for encoding the amino acid sequence of the present invention includes, in addition to the sequence disclosed in this specification, a sequence modified by the degenerate nature of the genetic code.

[0076] In the entirety of the following claims, the term “recombinant expression vector” collectively refers to a vector capable of expressing a peptide or protein encoded by a heterogeneous nucleic acid inserted into the vector, and preferably means having the form of an expression vector capable of expressing human glutathione synthase in the human body. In the entirety of the following claims, the term “vector” collectively refers to a DNA fragment, nucleic acid molecule, etc. delivered into a host cell, said vector can replicate DNA, be independently remanufactured in the host cell, and may be used interchangeably with the term “carrier.” And the term “expression vector” collectively refers to a recombinant DNA molecule that includes a target coding sequence and appropriate nucleic acid sequences essential for expressing the coding sequence operatively linked in a specific host organism. The term “operatively linked” collectively refers to a form in which the target coding sequence is linked in sequence with appropriate nucleic acid sequences in an expressible form, and there are no restrictions on the order and structure thereof. The above appropriate nucleic acid sequences are not limited thereto, but include, for example, promoter sequences that are transcription initiation factors to which RNA polymerase binds (e.g., CMV (cytomegalovirus) promoter for expression in eukaryotic cells, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha promoter, metallothionein promoter, beta-actin promoter, human IL-2 gene promoter, human IFN gene promoter, human IL-4 gene promoter, human lymphotoxin gene promoter, human GM-CSF gene promoter; It may include a tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL promoter, pR promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc. for expression in prokaryotic cells), any operator sequence for regulating transcription, an enhancer sequence for regulating expression levels, a start codon sequence coding for a suitable mRNA ribosome binding site, a stop codon sequence for regulating the termination of transcription and translation, a polyadenylation signal sequence (e.g., bovine growth hormone terminator, SV40-derived polyadenylation sequence, etc.), a selectivity marker sequence for selection (e.g., antibiotic resistance gene, drug resistance gene, nutritional requirement gene, etc.), and a tagging sequence that can easily isolate the expressed recombinant protein. As a vector, any commonly used carrier such as a plasmid, phage, cosmid, chromosome, or virus may be used, and preferably, it is an expression vector capable of expressing a target coding sequence in the human body. More preferably, it may include the nucleotide sequence represented by SEQ ID NO. 3. Additionally, variants of SEQ ID NO. 3 are included within the scope of the present invention. Specifically, it may include a nucleotide sequence having sequence homology of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more with the nucleotide sequence of SEQ ID NO. 3. However, it is not limited thereto as long as it is in a form capable of expressing glutathione synthase in the human body.And these recombinant vectors can be introduced into host cells using methods known in the art to transform the host cells. Examples of methods for introduction into host cells include, but are not limited to, the calcium chloride method, microprojectile bombardment, electroporation, PEG-mediated fusion, microinjection, and liposome-mediated method.

[0077] Including all claims below, "mitochondria" is a cellular organelle essential for the survival of eukaryotic cells involved in the synthesis and regulation of adenosine triphosphate (ATP) as an energy source. The mitochondria are associated with the control of various metabolic pathways in vivo, e.g., cell signaling, cell differentiation, and apoptosis, as well as the cell cycle and cell growth.

[0078] In the entirety of the following claims, "isolated or separated mitochondria" may mean mitochondria obtained from autologous, allogeneic, or xenogeneic sources.

[0079] In this specification, including all claims below, "autologous mitochondria" means mitochondria obtained from tissues or cells of the same individual. Additionally, the term "homologous mitochondria" means mitochondria obtained from tissues or cells of an individual that belongs to the same species as the individual but has a different genotype with respect to alleles. Additionally, the term "heterologous mitochondria" means mitochondria obtained from tissues or cells of an individual that belongs to a different species from the individual. In this case, the individual may be a mammal, and preferably a human.

[0080] Including all claims below, mitochondria may be isolated from tissues or cells of an organism. For example, the mitochondria may be obtained from somatic cells, germ cells, or stem cells, and may be isolated from blood cells or platelets. Most preferably, they are mitochondria isolated from mesenchymal stem cells overexpressing glutathione synthase.

[0081] In this specification, including all claims below, "cell" means a structural or functional unit constituting an organism, composed of cytoplasm surrounded by a cell membrane, and containing biomolecules such as proteins and nucleic acids. The cell means a cell containing mitochondria within a cell membrane.

[0082] In the entire specification including the following claims, "somatic cell" means a cell excluding germ cells among the cells constituting an organism. The somatic cell may be any one selected from the group consisting of muscle cells, liver cells, nerve cells, fibroblasts, epithelial cells, adipocytes, osteocytes, leukocytes, lymphocytes, platelets, mucosal cells, and combinations thereof.

[0083] In all claims below, the term "stem cell" in this specification refers to an undifferentiated cell capable of differentiating into various types of tissue cells. The stem cell may be any one selected from the group consisting of mesenchymal stem cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, bone marrow stem cells, neural stem cells, limbal stem cells, tissue-derived stem cells, and combinations thereof. In this case, the mesenchymal stem cell may be derived from any one selected from the group consisting of embryonic stem cells, umbilical cord, cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, placenta, synovial fluid, testis, periosteum, and combinations thereof. Preferably, it may be a mesenchymal stem cell derived from a human embryonic stem cell.

[0084] Additionally, the mitochondria may be obtained by concentrating and lysing cell or tissue samples isolated from an individual or cultured in vitro. Alternatively, the mitochondria may be obtained by lysing cell or tissue samples thawed after cryopreservation. Furthermore, the mitochondria may be obtained from freeze-dried, cryopreserved, or cell or tissue samples thawed after cryopreservation, but are not particularly limited as long as this does not have a negative effect on the biological activity of the obtained mitochondria.

[0085] In the entire specification including the following claims, the content of the mitochondria in the composition of the present invention can be appropriately adjusted according to the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., and, for example, may be 0.0001 to 99.9% by weight or 0.001 to 50% by weight based on the total weight of the composition, but is not limited thereto. The above content ratio is a value based on the dry weight after removing the solvent.

[0086] Including all claims below, the pharmaceutical composition according to the present invention may further comprise a suitable carrier, excipient, and diluent commonly used in the manufacture of pharmaceutical compositions. The excipient may be one or more selected from the group consisting of, for example, diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.

[0087] Including all claims below, the pharmaceutical compositions described herein may be used by being formulated in the form of external preparations such as powders, granules, sustained-release granules, enteric granules, liquids, ophthalmics, ellipsoids, emulsions, suspensions, ethanol tablets, troches, fragrances, limonene adrenergics, tablets, sustained-release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injectables, capsules, irrigation solutions, warning agents, lotions, pastes, sprays, inhalants, patches, sterile injectable solutions, or aerosols, according to conventional methods, and said external preparations may have formulations such as creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastes, or cataplasms. there is.

[0088] Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Other pharmaceutically acceptable carriers may be referenced from the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995). When formulating, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0089] Excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium monohydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, refined lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, primogel, etc., as additives to tablets, powders, granules, capsules, pills, and lozenges according to the present invention; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium casein, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch paste, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. may be used as binders, and hydroxypropylmethylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, anhydrous silica, 1-hydroxypropylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, D-sorbitol solution, hard anhydrous silica, etc. disintegrants;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium pods, kaolin, petroleum jelly, sodium stearate, cocoa paste, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silica, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and hard anhydrous silica may be used.

[0090] As additives to the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearic acid sucroses, polyoxyethylene sorbitol fatty acid esters (tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, water ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. may be used.

[0091] In the syrup preparation according to the present invention, a solution of white sugar, other sugars or sweeteners, etc. may be used, and if necessary, flavorings, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, viscosity enhancers, etc. may be used.

[0092] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0093] In the suspension agent according to the present invention, suspending agents such as acacia, tragacanthus, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910 may be used, and surfactants, preservatives, stabilizers, coloring agents, and fragrances may be used as needed.

[0094] The injectable preparation according to the present invention comprises solvents such as distilled water for injection, 0.9% sodium chloride injection solution, Ringer's injection solution, dextrose injection solution, dextrose + sodium chloride injection solution, PEG, lactated Ringer's injection solution, ethanol, propylene glycol, non-volatile oils—sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nijungtinamide, hexamine, and dimethylacetamide; and buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums. It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and sodium bisulfite acetone; non-inflammatory agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium CMC, sodium alginate, Tween 80, and aluminum monostearate.

[0095] The suppository according to the present invention comprises cocoa dough, lanolin, Witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, lanette wax, glycerol monostearate, Tween or Spandex, Imhausen, monollene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroccote SP, S-70-XXA, S-70-XX75 (S-70-XX95). Bases such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramount-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), and Tegestor triglyceride base (TG-95, MA, 57) may be used.

[0096] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the extract. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used.

[0097] Liquid preparations for oral administration include suspensions, oral liquids, emulsions, and syrups; in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0098] The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all the above-mentioned factors, and this can be easily determined by a person skilled in the art to which the present invention belongs.

[0099] The pharmaceutical composition of the present invention may be administered to an individual by various routes. All modes of administration are expected, for example, oral administration, subcutaneous injection, intraperitoneal administration, intramuscular injection, intrathecal (intradural) injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spray through the mouth or nose, skin administration, transdermal administration, etc.

[0100] The pharmaceutical composition of the present invention is determined by the type of active ingredient drug, along with various relevant factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and the severity of the disease. Specifically, the effective dose of the composition according to the present invention may vary depending on the patient's age, gender, and weight, and generally, 0.001 to 500 mg, preferably 0.01 to 100 mg per kg of body weight, may be administered daily or every other day, or divided into 1 to 3 doses per day. However, since the dose may be increased or decreased depending on the route of administration, the severity of the disease, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0101] Including all claims below, when mitochondria are used as food additives, the compound may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food or beverages, the mitochondria of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less, relative to the raw material. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the amount may be less than the above range, and since there are no issues regarding safety, the active ingredient may be used in an amount greater than the above range.

[0102] There are no specific restrictions on the types of the above-mentioned foods. Examples of foods to which the above-mentioned substance may be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense.

[0103] The health beverage composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as taumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrates is generally about 0.01-0.20g or about 0.04-0.10g per 100mL of the composition of the present invention.

[0104] In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, the composition of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. Although the proportion of these additives is not critical, it is generally selected in the range of 0.01 to 0.20 parts by weight per 100 parts by weight of the composition of the present invention.

[0105] The food composition of the present invention includes a health functional food composition.

[0106] In this specification, including all claims below, the term "health functional food" is synonymous with "food for special health use (FoSHU)" and refers to a food with high medical or health effects that is processed to efficiently exhibit bio-regulatory functions in addition to providing nutrition, and said food may be manufactured in various forms such as tablets, capsules, powders, granules, liquids, and pills to obtain useful effects for the prevention or improvement of diseases.

[0107] The health functional food of the present invention can be manufactured by methods commonly used in the industry, and can be manufactured by adding raw materials and ingredients commonly added in the industry. In addition, unlike general pharmaceuticals, it has the advantage of not having side effects that may occur from long-term use of pharmaceuticals because it is made of food, and it can be highly portable.

[0108] In this specification, including all claims below, the term “MitoTherapy” collectively refers to a pharmaceutical product used for therapeutic, diagnostic, and preventive purposes, comprising mitochondria produced by isolation, culture, and special manipulation from cells or tissues. Preferably, it refers to a therapeutic agent comprising mitochondria isolated from cells proliferated in vitro to restore the function of cells or tissues. The MitoTherapy of the present invention may additionally contain one or more active ingredients exhibiting the same or similar functions. Additionally, for administration, it may be further included by reference with a pharmaceutically acceptable carrier of the above pharmaceutical composition.

[0109] In the present specification, including all claims below, “antioxidant composition” is a pharmaceutical composition, food composition, or cosmetic composition for reducing oxidative stress in the body. More specifically, it means a composition that can be used to prevent or treat chronic inflammatory diseases (non-alcoholic fatty liver, non-alcoholic steatohepatitis, diabetes, diabetic complications, obesity, metabolic syndrome, etc.), autoimmune diseases (rheumatoid arthritis, inflammatory bowel disease, psoriasis, etc.), cancer cell growth, arteriosclerosis, etc. caused by reactive oxygen species by reducing “oxidative stress,” that is, in situations where harmful oxygen species increase in the body and cause or occur adverse effects on the human body.

[0110] Including all claims below, the “anti-inflammatory composition” in this specification is a pharmaceutical composition, food composition, or cosmetic composition for reducing inflammation in the body. More specifically, by reducing IL-6 (Interleukin-6), TNF-α (tumor necrosis factor-α), IL-1β, IL-18, etc., which induce inflammatory responses in the body, it [treats] chronic inflammatory diseases caused by inflammation (non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, diabetes, diabetic complications, obesity, metabolic syndrome, etc.), autoimmune diseases (rheumatoid arthritis, inflammatory bowel disease, psoriasis, etc.), respiratory inflammatory diseases (asthma, acute respiratory distress syndrome, inflammatory responses due to upper / lower respiratory infections, etc.), cardiovascular and vascular inflammatory diseases (atherosclerosis, vasculitis, myocarditis, etc.), neurological inflammatory diseases (Alzheimer's disease, Parkinson's disease, multiple sclerosis, neuropathic pain, encephalitis, etc.), kidney and liver inflammatory diseases (acute kidney injury, chronic renal failure, hepatitis, etc.), musculoskeletal inflammatory diseases (myositis, fasciitis, osteoarthritis, etc.), and skin and mucosal inflammatory diseases (atopic dermatitis, contact dermatitis, acne, It means a composition that can be used to prevent or treat conditions such as psoriasis, etc.

[0111] Including all claims below, the “pharmaceutical composition for the prevention or treatment of mitochondrial dysfunction” in this specification relates to chronic inflammatory diseases (non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, diabetes, diabetic complications, obesity, metabolic syndrome, etc.), autoimmune diseases (rheumatoid arthritis, inflammatory bowel disease, psoriasis, etc.), respiratory inflammatory diseases (asthma, acute respiratory distress syndrome, inflammatory response due to upper / lower respiratory tract infection, etc.), cardiovascular and vascular inflammatory diseases (atherosclerosis, vasculitis, myocarditis, etc.), neurological inflammatory diseases (Alzheimer's disease, Parkinson's disease, multiple sclerosis, neuropathic pain, encephalitis, etc.), kidney and liver inflammatory diseases (acute renal injury, chronic renal failure, hepatitis, etc.), musculoskeletal inflammatory diseases (myositis, fasciitis, osteoarthritis, etc.), skin and mucosal inflammatory diseases (atopic dermatitis, contact dermatitis, acne, psoriasis, etc.), and muscle and sarcopenia-related It refers to a composition that can be used to prevent or treat diseases (sarcopenia, muscle atrophy, mitochondrial myopathy, exercise intolerance, etc.) and aging-related diseases (immune aging, systemic inflammation, chronic inflammatory diseases, etc.). Additionally, it refers to a composition that can be used to treat cancer through the regulation of cancer metabolism based on mitochondrial stress.

[0112]

[0113] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0114]

[0115] [Example]

[0116] Example 1: Isolation of mitochondria

[0117] To prepare a glutathione synthetase (GS) expression vector, the GS gene (DNA sequence of SEQ ID NO. 1; amino acid sequence of SEQ ID NO. 2) was inserted into the pCMV6-AC Mammalian Expression Vector (OriGene Technologies, Inc.) to prepare a GS expression vector (CMV-GS plasmid, SEQ ID NO. 3).

[0118] And human embryonic stem cell-derived mesenchymal stem cells (hES-MSC, SETMCELL Technologies) TM ) 2X10 5 Dispense into a 6-well plate at the cell concentration, and 1% L-glutamine (Gibco TM Growth media (StemPro) supplemented with , 200mM) ® MSC SFM Xeno-free, Gibco TMThe cells were cultured for 24 hours at 37°C under 5% CO2 conditions. Subsequently, 500 ng of the prepared GS expression vector (GS plasmid) or a mock vector (mock plasmid) without the GS gene inserted was transfected using Lipofectamine™ Stem Transfection Reagent (ThermoFisher Scientific Inc.). Then, mitochondria were isolated from the transfected mesenchymal stem cells using a Mitochondria isolation kit (Thermo Scientific Inc.). Briefly, after culturing the mesenchymal stem cells again for 24 hours, the cells were obtained by centrifugation at 1,300 rpm for 3 minutes and then frozen at -80°C. Subsequently, 700 μL of separation reagent A was added and reacted on ice for 2 minutes, followed by the addition of 10 μL of separation reagent B and reacted again on ice for 20 minutes, while vortexing once every 5 minutes. Subsequently, 700 μL of separation reagent C was added and gently inverted to mix, followed by centrifugation at 700 g for 10 minutes at 4°C. Afterward, the supernatant (mitochondrial fraction) was transferred to a new 2 mL tube and centrifuged again at 12,000 g for 15 minutes at 4°C. The supernatant was then discarded, and 500 μL of separation reagent C was added to the precipitated mitochondrial pellet to resuspend it, followed by centrifugation again at 12,000 g for 5 minutes at 4°C. The supernatant was discarded, and the mitochondrial pellet was resuspended in phosphate buffered saline (PBS) to obtain mitochondria from the MSCs. The method for isolating mitochondria is briefly illustrated in Figure 1A, and the obtained mitochondria were stained with JC-1 staining reagent (Invitrogen TMBy measuring the mitochondrial membrane potential using ), it was confirmed whether healthy mitochondria were obtained. The results are shown in Figure 1B.

[0119] As shown in Fig. 1B, red fluorescence of JC-1 was detected, confirming that the JC-1 protein is bound in a polymeric form and exhibits a high membrane potential. Through this, it was confirmed that the isolated mitochondria were healthy and functionally active. Furthermore, analysis of the acquired mitochondria using a NanoSight analyzer revealed 2 x 10⁶ 5 100 μg of mitochondria were obtained from MSCs of cells, and it was confirmed that the particle size of the mitochondria was distributed in the range of 100 to 900 nm and the average diameter was about 392.1 nm.

[0120] Then, to determine the levels of glutathione (GSH) in the isolated mitochondria, MSCs transformed with a GS expression vector or a mock vector were seeded into 96-well plates and cultured for 2 days. Oxidative stress was induced in the cultured MSCs by treating them with 100 μM H2O2, and mitochondria were isolated after 1 day. Using the isolated mitochondria, the GSH / GSSG-Glo™ Assay Kit (Promega) was prepared according to the manufacturer's instructions. TMThe level of GSH was verified using (Corporation). Subsequently, all experiments were repeated at least three times, and the results were statistically analyzed using GraphPad Prism (GraphPad Software) and expressed as mean ± standard deviation. Statistical significance was confirmed using Student's t-test for comparisons between two groups and Analysis of Variance (ANOVA) for comparisons between three or more groups; a value of P < 0.05 was considered statistically significant. Hereinafter, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001. The results are shown in Figure 2.

[0121] As shown in Figure 2, it was confirmed that the concentration of GSH was higher in GS-mito (mitochondrials isolated from MSCs transformed with a GS expression vector) compared to Mock-mito (mitochondrials isolated from MSCs transformed with a Mock vector).

[0122] To determine the effect of oxidative stress on mitochondria, MSCs transformed with a GS expression vector or a mock vector were seeded into 96-well plates and cultured for 2 days. Oxidative stress was induced by treating with 1000 μM H2O2 for 4 hours, after which mitochondria were isolated. The mitochondrial membrane potential of the isolated mitochondria was measured using the JC-1 staining reagent. The results are shown in Figure 3A. The results of confirming the mitochondria using transmission electron microscopy (TEM) are shown in Figure 3B.

[0123] As shown in Figure 3A, in the case of GS-mito, it was confirmed that it maintained the JC-1 polymer form by exhibiting red fluorescence even under conditions where oxidative stress was induced, whereas in the case of Mock-mito, both the JC-1 polymer (red fluorescence inside the two dotted circles on the left in the bottom figure) and the JC-1 monomer (green fluorescence inside the two dotted circles on the right in the bottom figure) forms appeared due to oxidative stress.

[0124] In addition, as shown in Figure 3B, the results of TEM observation also confirmed that in the case of Mock-mito (left circle), many damaged mitochondria were observed, whereas in the case of GS-mito (right circle), the morphology of most normal mitochondria was observed.

[0125] Through the above results, it was confirmed that GS-mito exhibits high resistance to oxidative stress.

[0126]

[0127] Example 2: Confirmation of mitochondrial recovery efficacy against oxidative stress

[0128] To verify the efficacy of the isolated mitochondria, mouse myoblast C2C12 cells (ATCC, CRL-1772) were used. C2C12 cells were placed in a 6-well plate at a rate of 1 x 10⁶ 5 Cells were isolated, and 10% Fetal Bovine Serum (FBS, GIBCO₃) TM High glucose DMEM (GIBCO) added TMThey were cultured for 4 days in the medium. Subsequently, the medium was replaced with differentiation medium (High glucose DMEM supplemented with 2% inactivated Normal Horse Serum (NHS)) and cultured for 3 days to induce differentiation into myotubes. Oxidative stress was induced in C2C12 cells differentiated for 3 days by treating them with 200 μM H2O2. Then, after 1 day, 100 μg of mitochondria obtained by the same method as in Example 1 were added to the C2C12 cells, and the mitochondria were co-cultured via MitoCeption (Mitochondria Reception) and transplanted into the C2C12 cells. The co-cultured mitochondria undergo internalization through endocytosis or membrane fusion via the cell membrane. At this time, according to the manufacturer's instructions, the mitochondria of the C2C12 cells were [transplanted] into MitoTracker TM Two types of mitochondria were classified by staining with Deep Red FM probes (Invitrogen™) and mitochondria isolated from MSCs with MitoTracker™ Orange CMTMRos probes (Invitrogen™). After transplanting mitochondria and culturing for 3 days, immunostaining was performed to determine the effect of mitochondria on the recovery of muscle damaged by oxidative stress. Briefly, cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes and washed three times with cold PBS. Then, 0.05% Triton TMAfter permeabilization using PBS containing X-100, the cells were blocked using PBS containing 4% NHS at room temperature. Subsequently, Fast Myosin Heavy Chain (Fast MyHC) was stained primary using Anti-Fast Myosin Skeletal Heavy Chain Antibody (Abcam Limited) at 4°C for 16 hours. After removing all unbound antibodies through three washes, secondary staining was performed using a secondary antibody and DAPI at room temperature for 1 hour. The cells were then observed using a Zeiss LSM 710 confocal microscope. The experimental procedure is briefly outlined in Fig. 4A, and the results of confirming the mitochondria transplanted into the cells are shown in Fig. 4B. The results of confirming the myoducts are shown in Figs. 5, 6A, and 6B.

[0129] As shown in Figure 4B, it was confirmed that mitochondria (white) isolated from MSCs were internalized into C2C12 24 hours after transplantation, confirming that mitochondria were successfully transplanted into the cell.

[0130] As shown in Figure 5, it was confirmed that the number of myocanopies decreased in both oxidative stress-induced C2C12 cells and C2C12 cells transplanted with oxidative stress-induced Mock-mito, whereas the number of myocanopies increased in C2C12 cells transplanted with GS-mito.

[0131] In addition, as shown in Figures 6A and 6B, when comparing the green fluorescence of Fast MyHC and the blue fluorescence of the nucleus, C2C12 transplanted with Mock-mito showed the form of an early, immature myotube (nascent myotube) with clustered nuclei, whereas C2C12 transplanted with GS-mito showed a form of mature myotube with nuclei aligned in a straight line and increased internuclear distance.

[0132] And using the acquired fluorescence image, 1.3 mm 2 Number of root canals contained within, 1.3mm 2 % of the total area of ​​Fast MyHC positive (+) contained within, 1.3mm 2 The fusion index (%) and the ratio of length (mm) to width (mm) of the root canal were quantified using Image J and are shown in Figures 7A to 7D, respectively. Values ​​were extracted from at least three images for each group.

[0133] As shown in Figures 7A and 7B, the number of root canals per unit area and the area of ​​Fast MyHC per unit area were significantly increased in the GS-mito experimental group compared to other experimental groups, and it was confirmed that they increased to a level similar to that of the normal control group that was not subjected to oxidative stress. As shown in Figure 7C, the fusion index also increased significantly in the GS-mito experimental group, and as shown in Figure 7D, it was confirmed that the ratio of length to width of the root canal also increased to a level similar to that of the normal control group.

[0134] Through the above results, it was confirmed that the GS-mito of the present invention promoted the formation of C2C12 cells even in muscle damage caused by oxidative stress, and exhibited stronger recovery power even in high concentrations of H₂O₂.

[0135]

[0136] Example 3: Confirmation of mitochondrial function in transplanted cells

[0137] To confirm the function of GS-mito in transplanted cells, C2C12 cells were cultured for 3 days in the same manner as in Example 2, then replaced with differentiation medium and cultured for 4 days to differentiate into myotubes. Subsequently, oxidative stress was induced in the differentiated C2C12 cells by treating them with 200 μM H2O2, and after 1 day, 100 μg of isolated mitochondria were added to the C2C12 cells in the same manner as in Example 1 and cultured to transplant the mitochondria into the C2C12 cells. After culturing for one week, the Oxygen Consumption Rate (OCR) was measured according to the manufacturer's instructions using a Seahorse XF Analyzer (Agilent Technologies) and a Seahorse XF Cell Mito Stress Test Kit (Agilent). Figure 8A briefly illustrates the experimental method, and Figure 8B shows the results of microscopic observation of the morphology of the myotubes transplanted with mitochondria. Figures 8D and 8E show the measured OCR results.

[0138] As shown in Figures 8D and 8E, it was confirmed that cells transplanted with GS-mito showed significantly increased maximal respiration induced by FCCP (Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) and spare respiratory capacity induced by Rotenone and Antimycin A compared to cells transplanted with Mock-mito. High maximal respiration implies that cells transplanted with GS-mito possess a higher potential for energy production under conditions of high energy demand and have improved mitochondrial enzyme activity. Spare respiratory capacity is defined as the difference between basal respiration and maximal respiration, and high spare respiratory capacity implies that the cells possess mitochondrial functional adaptability, which allows them to adapt more flexibly to sudden increases in energy demand within the cell, as they have a high amount of additional energy reserves available for mobilization during stress or increased energy demand.

[0139] Through the above results, it was confirmed that the GS-mito of the present invention transplanted cells had improved mitochondrial energy production capacity and function, and possessed high resilience and viability under stressful conditions.

[0140]

[0141] Example 4: Confirmation of therapeutic effect in an animal model with induced sarcopenia

[0142] To confirm whether the GS-mito of the present invention is effective in muscle regeneration in vivo, experiments were conducted using 37-week-old BALB / c mice (Coatech Co., Ltd.) with an average body weight of approximately 30g. All animal experiments below were conducted with the approval of the Institutional Animal Care and Use Committee (SNUH-IACUC) of Seoul National University Hospital. More specifically, three 37-week-old BALB / c mice, a general aging model; three mice in which muscle atrophy was induced by immobilizing one of the hind legs into an e-tube (immobilization; IM) to prevent leg use; three mice in which PBS was injected at the same dose as the experimental group into three sites of the gastrocnemius muscle one day after remobilization (RM) following leg immobilization for two weeks; three mice in which 100 μg of Mock-mito was injected into three sites of the gastrocnemius muscle one day after remobilization following leg immobilization for two weeks; three mice in which 100 μg of GS-mito was injected into three sites of the gastrocnemius muscle one day after remobilization following leg immobilization for two weeks; and 1X10⁶ mice transformed with a Mock vector into three sites of the gastrocnemius muscle one day after remobilization following leg immobilization for two weeks. 5 Two mice injected with MSCs of cells, and 1 x 10⁶ cells transformed with a GS expression vector into three sites of the gastrocnemius muscle one day after releasing the leg fixation following 2 weeks of leg immobilization. 5 The experiment was conducted by dividing mice into two groups injected with MSCs from the cells. Considering the amount of mitochondria lost during extraction, 2 x 10 5 When converted to the amount of mitochondria extracted from the MSCs of the cells, 1 x 10⁶ 5 It is predicted that the MSCs of the cells contain more than 100 μg of mitochondria, so the amount of MSCs is 1 x 10⁻¹⁰ 5Experiments were conducted using cells. Mitochondria or MSCs were injected, and a motion test was performed 13 days later. One day after the motion test, the subjects were euthanized, and blood samples were collected from the heart for serum biochemical analysis. For muscle experiments, the gastrocnemius and soleus muscles were obtained from the fixed hind leg and the opposite unfixed hind leg, respectively, for the subsequent experiments. Figure 9 briefly illustrates the experimental method. The weight of the gastrocnemius muscle in the sarcopenia-induced leg was divided by the weight of the gastrocnemius muscle in the opposite leg without sarcopenia to express the increase or decrease in muscle weight as a percentage (%). The results are shown in Figure 10. The maximum muscle strength of each mouse was determined by measuring five consecutive times without rest using a Grip Strength Meter (Columbus Instruments International Corporation). The measured maximum muscle strength value (g) was then normalized by dividing it by body weight (g) to calculate the muscle force / body weight ratio. The results are shown in Fig. 11. The acquired gastrocnemius and soleus muscles were observed using TEM. For TEM observation, the acquired tissues were fixed in a 2.5% glutaraldehyde solution dissolved in PBS (pH 7.4) at 4°C for 72 hours, followed by washing with PBS. Subsequently, they were post-fixed in a 2% osmium tetroxide solution on ice for 1 hour, followed by stepwise dehydration using ethanol and propylene oxide, and then embedded in Epon 812 resin. Resin polymerization was carried out with pure resin at 70°C.The results of observing the prepared tissue with TEM are shown in Figure 12.

[0143] As shown in Figure 10, mice injected with Mock-mito or MSC showed some increase in muscle weight compared to mice injected with PBS, but mice injected with GS-mito showed an increase in muscle weight similar to the normal control group, confirming that damaged muscles can be effectively regenerated.

[0144] As shown in Figure 11, in the case of muscle strength, it was confirmed that mice injected with GS-mito showed increased muscle strength similar to the normal control group, and through this, it was confirmed that muscle function was significantly improved.

[0145] As shown in Figure 12, in a sarcopenia model (IR+RM muscle) in which muscle atrophy and sarcopenia are induced by fixing the hind legs, damaged mitochondrial morphology is observed, but in mice injected with GS-mito, it was confirmed that the mitochondrial morphology is similar to that of normal mice.

[0146] Through the above results, it was confirmed that the GS-mito of the present invention is effective in normalizing muscle function by promoting the regeneration and increase of damaged or reduced muscles in a sarcopenia model.

[0147]

[0148] Example 5: Confirmation of therapeutic effects according to mitochondrial concentration in an animal model of sarcopenia-induced disease

[0149] To confirm the therapeutic effect according to the concentration of mitochondria, an experiment was conducted using 50 μg and 100 μg of mitochondria in the same manner as in Example 4. The experimental results confirming the grip strength of mice are shown in Figure 13.

[0150] As shown in the left diagram of Fig. 13, muscle strength increased in the experimental group injected with 50 μg of GS-mito compared to the experimental group injected with Mock-mito, but as shown in the right diagram of Fig. 13, it was confirmed that muscle strength recovered to a level similar to that of normal mice in the experimental group injected with 100 μg of GS-mito.

[0151] In addition, to compare the therapeutic effects of the cell therapy agent (MSC) and mitochondria at different concentrations, the experiment was conducted in the same manner as in Example 4. The results of measuring muscle weight are shown in Fig. 14, and the results of measuring grip strength are shown in Fig. 15. The number of mice for each experimental group is indicated in the respective figures.

[0152] As shown in Figure 14, mice injected with MSCs transformed with a GS expression vector showed a slight increase in muscle weight compared to mice injected with MSCs transformed with a Mock vector, which was similar to the increase in muscle weight of mice injected with 50 μg of GS-mito. It was also confirmed that mice injected with 100 μg of GS-mito showed a muscle weight similar to that of normal mice.

[0153] As shown in Figure 15, in the case of grip strength, MSCs transformed with a GS expression vector and mice injected with 50 μg of GS-mito showed similar muscle strength recovery, and mice injected with 100 μg of GS-mito showed muscle strength similar to that of normal mice.

[0154] In addition, the level of glutathione was measured using the obtained mouse serum in the same manner as in Example 1. The results are shown in Fig. 16.

[0155] As shown in Figure 16, it was confirmed that glutathione concentration was significantly increased in mice injected with 100 μg of GS-mito.

[0156] In addition, the expression level of 4-Hydroxynonenal (4HNE) was confirmed through Western blotting. More specifically, proteins were extracted from the acquired gastrocnemius muscle tissue, denatured by heating at 95°C for 5 minutes, and then separated by size using a 10% SDS-polyacrylamide gel. The separated proteins were then transferred to an NC membrane and blocked with 5% skim milk for 1 hour. Subsequently, anti-4 Hydroxynonenal antibody (Abcam Limited) or anti-HSC70 antibody (Abcam Limited) was added as the primary antibody and reacted for 16 hours, after which all unbound antibodies were removed by washing three times with PBS. Then, a secondary antibody reaction was performed by adding anti-rabbit IgG or anti-mouse IgG conjugated with horseradish peroxidase (HRP), and detection was performed using Electrochemiluminescence (ECL). Each band was then quantified using Image J and normalized to the HSC70 value. The results are shown in Figure 17.

[0157] As shown in Figure 17, it was confirmed that 4HNE was most effectively reduced in mice administered GS-mito.

[0158] In addition, to determine whether it reduces inflammation, mouse blood was obtained and the amount of IL-6 was measured using a Mouse IL-6 ELISA kit (Thermo Fisher Scientific Inc.). The results are shown in Figure 18.

[0159] As shown in Figure 18, it was confirmed that inflammation was most effectively reduced in mice administered GS-mito.

[0160] Through the above results, it was confirmed that the GS-mito of the present invention can effectively promote the regeneration of damaged or reduced muscle in vivo depending on the concentration, thereby restoring muscle function. Furthermore, the increase in glutathione concentration and the decrease in 4NHE in the tissue confirmed that GS-mito exhibits a strong antioxidant effect in the transplanted tissue and can improve redox balance by restoring the function of damaged mitochondria to normal levels. Additionally, it was confirmed that the GS-mito of the present invention can also effectively reduce inflammation through its anti-inflammatory ability.

[0161]

[0162] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0163] Since the mitochondria according to the present invention, or the mitochondrial transplant therapeutic agent containing the same, or the composition containing the same utilizes mitochondria rather than living cells, it can not only reduce the general side effects associated with cell therapies but also enable low-cost mass production by lowering regulatory barriers and significantly reducing production unit costs. Furthermore, because the mitochondria according to the present invention can treat muscle loss through various mechanisms—such as increasing resistance to oxidative stress in transplanted cells, reducing inflammation, enhancing mitochondrial function, and promoting the regeneration of damaged or diminished muscles to increase muscle mass and improve muscle function—it can be widely applied to various diseases related to muscle loss.

Claims

1. Cell-derived mitochondria overexpressing glutathione synthetase.

2. In claim 1, the mitochondria satisfies one or more of the following characteristics: i) Indicates high glutathione concentration; ii) Indicates a high membrane potential; iii) exhibits high resistance to oxidative stress; and iv) Increase muscle mass.

3. In claim 1, the cell overexpressing the glutathione synthase is a mitochondrion that is a cell transformed with a recombinant expression vector comprising a promoter and a polynucleotide encoding glutathione operably linked thereto.

4. In paragraph 1, the cell is a somatic cell or a stem cell, a mitochondria.

5. In paragraph 4, the stem cells are mitochondria selected from the group consisting of mesenchymal stem cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, and induced pluripotent stem cells.

6. A mitochondrial transplant therapy (MitoTherapy) for the prevention or treatment of muscle weakness-related diseases, comprising the mitochondria of claim 1 as an active ingredient.

7. In paragraph 6, the above-mentioned muscle weakness-related disease is a disease caused by mitochondrial dysfunction, a mitochondrial transplant therapy.

8. In claim 6, the mitochondrial transplant therapy, wherein the muscle weakness-related disease is one or more selected from the group consisting of sarcopenia, muscular atrophy, muscle dystrophy, cardiac atrophy, mitochondrial myopathy, amyotrophic lateral sclerosis (ALS), cardiomyopathy, Parkinson's disease, Huntington's disease, Alzheimer's disease, and peripheral neuropathy.

9. A pharmaceutical composition for the prevention or treatment of diseases related to muscle weakness, comprising the mitochondria of claim 1 as an active ingredient.

10. A pharmaceutical composition according to claim 9, wherein the muscle weakness-related disease is a disease caused by mitochondrial dysfunction.

11. A pharmaceutical composition according to claim 9, wherein the muscle weakness-related disease is one or more selected from the group consisting of sarcopenia, muscular atrophy, muscle dystrophy, cardiac atrophy, mitochondrial myopathy, amyotrophic lateral sclerosis (ALS), cardiomyopathy, Parkinson's disease, Huntington's disease, Alzheimer's disease, and peripheral neuropathy.

12. A food composition for the prevention or improvement of diseases related to muscle weakness, comprising the mitochondria of claim 1 as an active ingredient.

13. A food composition according to Clause 12, wherein the above-mentioned muscle weakness-related disease is a disease caused by mitochondrial dysfunction.

14. A food composition according to claim 12, wherein the above-mentioned muscle weakness-related disease is one or more selected from the group consisting of sarcopenia, muscular atrophy, muscle dystrophy, cardiac atrophy, mitochondrial myopathy, amyotrophic lateral sclerosis (ALS), cardiomyopathy, Parkinson's disease, Huntington's disease, Alzheimer's disease, and peripheral neuropathy.

15. An antioxidant composition comprising the mitochondria of claim 1 as an active ingredient.

16. An anti-inflammatory composition comprising the mitochondria of claim 1 as an active ingredient.

17. A pharmaceutical composition for the prevention or treatment of mitochondrial dysfunction, comprising the mitochondria of claim 1 as an active ingredient.

18. A method for preventing or treating a muscle weakness-related disease comprising the step of administering a composition containing the mitochondria of claim 1 as an active ingredient in a therapeutically effective amount to an individual in need thereof.

19. Use of a composition comprising the mitochondria of claim 1 as an active ingredient for the prevention, improvement, or treatment of diseases related to muscle weakness.

20. Use of a composition containing the mitochondria of claim 1 as an active ingredient for the production of a drug for the prevention, improvement, or treatment of diseases related to muscle weakness.

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